We have created an allelic PTHrP-lacZ knockin mouse that provides a simple and sensitive reporter of PTHrP gene expression. The PTHrP-lacZ mouse demonstrates PTHrP gene expression in a wide variety of locations, including several that were previously unappreciated or underappreciated. These include: 1) the insertion sites of tendons and ligaments into cortical bone (collectively referred to as entheses), 2) the periosteum itself, particularly during bone growth, and 3) superficial layers of articular cartilage. In all of these sites, the PTHrP gene seems to be mechanically-induced. We have not identified PTHrP/lacZ expression in any internal bone cell population. We propose that PTHrP functions "at the gates" of endochondral bones, namely, at the interface of skeletal elements with each other and with surrounding muscles and connective tissues. Aim 1. We will characterize PTHrP, PPR, and cell marker expression in selected periosteal sites and entheses in order to: a) identify the cells that participate in paracrine signaling in these sites and b) enable the functional experiments proposed in Aims 2 and 3. We have also bred a single lacZ allele-containing PTHrP-null mouse that serves as a reporter system for Indian hedgehog regulation of PTHrP gene expression. Aim 2 comprises two overlapping subaims. One involves mechanical regulation of PTHrP expression and chondrocyte or bone cell function(s) in the periosteum, fibrocartilage, and fibrous entheses. The second considers specific candidate PTHrP functions in the periosteum, including a) mediation of force-driven periosteal bone formation, b) regulation of the modeling of the metaphysis and diaphysis during linear growth, and c) potential PTHrP involvement in the sexual dimorphism of periosteal bone. Aim 3. We will employ the scleraxis and/or periostin genes in order to create mouse models in which PTHrP is conditionally deleted in the periosteum and entheses. PUBLIC HEALTH RELEVANCE. Most bones develop from an initial cartilagenous mold. Such bones are comprised of two separate compartments: internal (or endosteal) bone and external (or periosteal) bone. Endosteal bone evolved principally to subserve reproductive demands and is primarily regulated by estrogen. Periosteal bone accounts for the larger and stronger skeleton in the male and is regulated primarily by androgens and mechanical forces (and inhibited by estrogen). We have identified parathyroid hormone-related protein (PTHrP) in the periosteum and ligament and tendon insertions into periosteal bone and also in articular cartilage the joints. In all of these sites, PTHrP production is regulated by mechanical force. We propose that PTHrP regulates cartilage and bone cell functions "at the gates" on long bones, namely, at the interface of the bones with each other and with surrounding muscles and connective tissues. Examples of such functions include PTHrP mediation of the shaping of bones by mechanical force, the sculpting of bones as they grow, and the sex steroid modulation of skeletal size.